Abstract Critical to the future progress of cancer immunotherapies is the discovery and tracking of T cells specific for cancer cells but not normal tissues. Neo-antigen derived from mutations are a group of attractive antigens for cancer immunotherapy. Because they are mutated variants of self-antigens, they are immunologically considered as foreign-antigens and are not part of the self-antigens. Thus, these cells are able to retain high affinity T cell receptor (TCRs) bearing cells that are more cytotoxic T cells. Neo-antigen based therapies, whether as neo-antigen vaccines or using neo-antigen specific T cell receptor (TCR) sequences in TCR re-directed adoptive cell transfer therapy, have become very promising in the fight against cancer. However, one potential complication of neo-antigen based therapies is the cross-reactivity of neo-antigen activated T cells might have toward the wildtype counterpart peptide (WT peptide). Therefore, high-throughput screening for neo-antigens that only induce neo-antigen specific T cell responses or high-throughput screening for neo-antigen only TCRs is critical for the wide application of neo-antigen based cancer immunotherapy approach in clinical settings. However, current technology does not permit a quick screening for none-WT reactive neo-antigens or none-WT reactive neo-antigen-specific T cells from hundreds of neo-antigen candidates. Using a recently developed technology that links TCR specificities to TCR sequences at single cell level in a high-throughput manner, we aim to develop a high-throughput platform for screening non-cross-reactive neo-antigens and neo-antigen specific TCRs for cancer immunotherapy.